Automated staining and decolorization of biological material

ABSTRACT

An improved method and apparatus for staining samples of biological material for accurate analysis of the sample. Biological material is applied to a substrate, such as a microscope slide. The biological specimen then is then stained with a selected staining composition, which may be gentian violet for a Gram&#39;s Stain analysis. The stained biological material is at least partially decolorized and the level of decolorization is analyzed optically. If necessary, the decolorizing step and the optical analysis steps are repeated until a selected level of decolorization is obtained.

1. CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. ProvisionalApplication Serial No. 60/116,485, filed Jan. 20, 1999, entitledAUTOMATED STAINING AND DECOLORIZATION OF BIOLOGICAL MATERIAL.

BACKGROUND OF THE INVENTION

[0002] 2. Field of the Invention

[0003] The present invention relates generally to methods and apparatusfor the diagnostic staining of biological material. More particularly,the present invention relates to methods and apparatus for stainingbiological material on microscope slides in an automated fashion.

[0004] 3. Background and Summary of Prior Art

[0005] Biological material, whether viruses, bacteria, or various smearsor samples of blood, mucus, and the like, have been analyzed forcenturies, since Van Leeuvenhoek invented the microscope. Typically thesample of material is applied to a microscope slide, stained orotherwise rendered into an analyzable state, and analyzed by a humantechnician or scientist.

[0006] As laboratory services for hospitals, physician's offices,veterinarians, and other life-science-based enterprises becomesincreasingly “outsourced,” the laboratory's ability to analyze slides ofbiological material rapidly and accurately becomes increasinglyimportant. Several steps have been made toward automating theseprocesses, but it seems unlikely that the skilled human technician willbe almost entirely removed from the process in the foreseeable future.

[0007] A fairly typical and important laboratory staining technique isknown as Gram's Stain, which was devised by H. C. J. Gram. The Gram'sStain is a “gateway” test that indicates to the technician the presence(or absence) of certain bacteria in a sample of biological material andgives the technician or scientist information necessary or helpful tomake further analysis. For instance, Gram's Stain can be used todetermine which pathogens are suspected and lead to an antibioticprescription until further identification can be conducted.

[0008] Like several staining or analysis methods, Gram's Stain involvestreating the biological material, usually applied to a conventionalmicroscope slide, with a number of reagents or stains. The reagents orstains emphasize or highlight the presence (or absence) of certain typesor features of bacteria or other biological material that is helpful tothe technician. In the Gram's Stain, the bacteria are treated first withgentian violet, and then with a formulation of iodine conventionallyknown as Gram's iodine. This stains almost all of the bacteria a deepblue or violet. “Gram positive” bacteria absorb the gentian violet andGram's iodine into their cellular structure, while “Gram negative”bacteria are stained only superficially. The sample is then washed withacid alcohol, which “decolorizes” or washes the color from Gram negativebacteria. Thus, when adequate decolorization has occurred, the blue orviolet Gram positive bacteria can be distinguished from the colorless(or less deeply blue or violet) Gram negative bacteria. A“counter-stain,” of fuchsine for example, may be applied to turn theblue or violet bacteria to a reddish shade to improve their visibility.Decolorization is critical to the Gram's Stain because too littledecolorization can yield false Gram positives and too muchdecolorization can yield false Gram negatives.

[0009] Several past attempts at providing automating the slidepreparation and staining process have met with limited success. Forexample, U.S. Pat. No. 4,029,470, Jun. 14, 1977 to Wilkins et al.provides an apparatus for automatically staining a single microscopeslide without a lab technician touching the slide. This patent addressesthe decolorization issue by timing the application of decolorizing agentin selected volume. The time/volume control of decolorization isinsufficient to accurately decolorize a Gram Stain. The decolorizationprocess is simply too dependent upon observation and manual work to beso easily controlled.

[0010] GG&B Technology, Inc., of Wichita Falls, Tex., sells a moresophisticated slide stainer under the registered trademark Quick Slide®.This device fully automates the preparation of slides for analysis andis a useful tool in the modern medical laboratory. Nevertheless, theQuick Slide® device is not capable of accurately decolorizing slides fora staining process such as the Gram's Stain.

[0011] U.S. Pat. Nos. 5,545,535; 4,665,024; and 4,639,421 all discloseflourescent gram stains and methods of analyzing bacteria stained withthe flourescent dyes in which a spectral analysis of the fluorescence ofthe stained bacteria is used to analyze the Gram positive and negativebacteria in the sample. These inventions do not address thedecolorization issue because it seems that decolorization is lessimportant (or unimportant) where flourescent dyes or stains are used inlieu of the conventional Gram's Stain of gentian violet.

[0012] A need exists, therefore, for an automated method and apparatusfor staining biological material and accurately decolorizing the stainedsample prior to analysis.

SUMMARY OF THE INVENTION

[0013] It is a general object of the present invention to provide animproved method and apparatus for staining samples of biologicalmaterial for accurate analysis of the sample. These and other objects ofthe invention are achieved by applying the biological material to asubstrate, preferably a microscope slide. The biological material thenis then stained with a selected staining composition, which may begentian violet for a Gram's Stain analysis. The stained biologicalmaterial is at least partially decolorized and the level ofdecolorization is analyzed optically. If necessary, the decolorizingstep and the optical analysis steps are repeated until a selected levelof decolorization is obtained.

[0014] Other objects, features, and advantages of the present inventionwill become apparent with reference to the drawings and description,which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a schematic depiction of the apparatus according to thepresent invention.

[0016]FIG. 2 is a high-level flow chart of the method according to thepresent invention.

[0017]FIG. 3 is a schematic depiction of the decolorization analysismodule of the apparatus of FIG. 1.

[0018]FIG. 4 is a more detailed schematic description of an apparatusfor staining biological materials according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Referring now to the figures, and particularly to FIG. 1, ahigh-level flowchart depicts the steps of the method or processaccording to the present invention. At block 11, a sample of biologicalmaterial is applied to the substrate, typically a conventionalmicroscope slide, to create a specimen. The biological specimen can beaffixed or applied in a number of conventional ways, which dependlargely on preference and subsequent processing. The biological specimenmay include blood, mucus, tissue samples, or the like, and is obtainedin conventional ways.

[0020] At step 13, the biological specimen is stained or dyed accordingto the analytical process to be employed. For the Gram Stain, the dye orstain is crystal or gentian violet in combination with Gram's iodine.The stain or dye stains the components of the biological specimen, insome cases only superficially; in others, the dye is absorbed within thecell structure.

[0021] At step 15, the biological specimen is decolorized, that is, thedye or stain that is not absorbed within the cell structure, but onlysuperficially, is washed away. For the Gram Stain, the decolorization isaccomplished by washing the specimen with acid alcohol (alcohol treatedto be slightly acidic). The washing may be controlled by volume of fluidapplied and time of application to yield a very rough measure of thelevel of decolorization achieved in this step.

[0022] At step 17, the level of decolorization is measured or analyzed.The specific method of analysis employed is discussed in greater detailwith reference to FIG. 3. Generally, the method applies radiation(visible spectrum, infrared, ultraviolet) to the “run-off” fluid ormaterial resulting from application of acid alcohol to the specimen. Theradiation passes through the acid alcohol, which itself is stained withgentian violet, Gram's iodine, and a safrin counter-stain. Because therun-off fluid is stained, the it absorbs certain wavelengths orfrequencies of the radiation and transmits others. The radiationtransmitted through the run off fluid is measured and compared to athreshold value. The threshold value is empirically determined basedupon the characteristics of the staining and decolorization process used(e.g. dye or stain characteristics and composition and decolorizingagent characteristics and composition).

[0023] At step 19, the comparison is analyzed to determine if thedesired or selected level of decolorization has occurred. If so, theprocess ends, at step 21, and the slide is ready for further processingor analysis. If the level of decolorization is inadequate, more washingwith decolorant occurs, as well as the analysis of step 17, until thesample is adequately decolorized.

[0024]FIG. 2 is a schematic depiction of the apparatus employing theprocess of FIG. 1. The apparatus comprises a slide carrier or mount 31on which the microscope slide or other specimen substrate is placed orcarried. A collector 33 is provided to collect or catch process fluidsas they run off the slide. The apparatus is also provided with fivereservoirs, 35, 37, 39, 41, 43 for the process fluids, which, for theGram Stain process, include crystal or gentian violet, Gram's Iodine,safrin (counter-stain), acid alcohol, and water. These Reservoirs areconnected to a fluid handling and metering system 45 that washes thebiological specimen with the appropriate fluid in the appropriateamount. Time- and volume-controlled application of the staining fluids(violet, iodine, and safrin) is adequate for those fluids to performtheir function. As previously discussed, time and volume control of thedecolorization process, alone, is not adequate.

[0025]FIG. 3 is a schematic representation of the “decolorizationmodule” of the apparatus of FIG. 2. The decolorization module can bemanufactured as part of the slide carrier or mount (31 in FIG. 2), orcan be a physically separate portion of the apparatus. The run-off fluidfrom the decolorization process, which includes acid alcohol, eitherdiluted with water or not, is collected by collector 33. The run-offfrom the decolorization step is collected in a chamber 51, which ispreferably optically transparent. In addition to chamber 51, thedecolorization module includes a source of radiation 53 arranged andlocated to irradiate a the fluid contents of chamber 51 with radiationof a selected wavelength or combination of wavelengths.

[0026] The run-off fluid, being stained, will transmit certainwavelengths of radiation and absorb others. The radiation transmittedthrough chamber 51 and run off fluid impinges on a photo detector 55.Photo detector 55 and associated electrical circuitry, including filtersand the like, generates an electrical signal whose strength isproportional to the intensity and wavelength of the radiation impingingon detector 55. This signal is input to a comparator or similar devicecapable of comparing the signal to a pre-determined threshold valuerepresentative of adequate decolorization. If the signal comparesfavorably with the threshold value, the user is alerted and the stainedslide is indicated to be ready for visual analysis. If the measuredlevel of decolorization indicates that decolorization is inadequate, thespecimen can be washed again and the run-off fluid analyzed as set forthabove.

[0027] The transmissibility of radiation through the run off fluid 55 isa function of the level of decolorization of the specimen on the slide.Thus, the level of decolorization can be empirically determined tocorrespond to a threshold value of transmitted radiation detected byphoto detector 55. Thus, the proper level of decolorization is opticallydetected without human interference or involvement, which permitsaccurate, automated slide staining to a degree not previously available.

[0028]FIG. 4 is a more detailed schematic of the apparatus according tothe present invention. Central to the apparatus is the control systemresiding on a personal computer (PC) 111, which may be a PC dedicatedfor use with the invention, or a conventional PC with control softwareresident on the harddrive and RAM, for use with the invention as needed.The other components, which may be external to the PC, are housed withinan enclosure (not shown) that is coupled to the PC.

[0029] The enclosure includes reservoirs (not shown) for the processfluids, which include, for Gram staining, gentian violet, Gram's iodine,distilled water, safrin, and acid alcohol. Other fluids may beappropriate for other staining processes. An EEPROM 113, programmedappropriately, is coupled to the computer through an IEEE 488 interface,and also to a series of stepper motors 115. Together, EEPROM 113 andstepper motors 115 control the flow of process fluids from thereservoirs, through fluid passages, to the microscope slide. The run-offof decolorization fluid (acid alcohol), is captured temporarily in aglass cuvette 121. EEPROM 113 is an appropriately programmedBell-Milgray 93LC46/P. Stepper motors 115 and their associatedcontrollers areOriental Motors Model PK264-01A, and Newark NDN2878U,respectively.

[0030] Adjacent the microscope slide, a regulated power supply powers alight source 123, which is a Gilway 5-Volt, 775-Ampere technical lamp.This is the radiation source and is arranged to direct visible-spectrumradiation through the cuvette and its contents. A Digi-Key OPT202P-NDphotodiode 125 with an on-chip amplifier is arranged on the side ofcuvette 121 opposite the lamp. A wavelength-selective filter 127, OmegaOptical 200 BP25, is interposed between the run-off fluid in the cuvetteand photodiode 125, and absorbs or filters radiation from the lamp 123outside the spectral range of about 200 to 340 nanometers, which permitspassage of the violet portion of the visible spectrum. The violetspectral components passed by filter 127 are absorbed (or not) by therun-off or decolorizing fluid in the cuvette and the total passedradiation impinges on the photodiode 125. The use of filter 127 reducesthe number of wavelengths of radiation impinging on photodetector 125and reduces the likelihood of error in the detection of the amount ofradiation passing through the run-off fluid, which is indicative of thelevel of decolorization.

[0031] The electrical signal output of photodiode 125, which isproportional to the level of decolorization of the slide, is routedthrough an Analog Devices AD976AAN analog-to-digital converter 131. Thedigital output of converter then is input to the computer for analysisby a comparison routine. The comparison routine may compare the outputof the photodiode to a discrete, empirically determined threshold, ormay use a variety of conventional, but more sophisticated, analysistechniques to determine whether the stained sample is appropriatelydecolorized or whether more decolorization (acid alcohol rinse) iswarranted. The PC can display various information about the stainingprocess, including process fluid levels, number of slides processed,decolorization level, calibration data, and the like. Theabove-described embodiment is by way of example of a staining apparatusaccording to the present invention for Gram staining, it is not intendedto be limiting of the invention.

[0032] The present invention provides a number of advantages. Primarily,it permits automation of the preparation of stained slides when thestaining process involves decolorization, as do many slide stainingprocedures, including the Gram Stain. The method and apparatus accordingto the present invention are sufficiently simple to be reliable andrelatively inexpensive to produce.

[0033] The invention has been described with reference to a preferredembodiment thereof. It is thus not limited, but is susceptible tovariation and modification without departing from the scope of theclaims.

1. A method of staining and analyzing biological specimen comprising thesteps of: applying the biological specimen to a substrate; staining thebiological specimen with a selected staining composition; at leastpartially decolorizing the biological specimen; optically detecting alevel of decolorization; and repeating, if necessary, the decolorizingstep and the optically detecting step until a selected level ofdecolorization is obtained.
 2. The method according to claim 1 whereinthe staining composition is a Gram's stain composition comprisinggentian violet and Gram's iodine.
 3. The method according to claim 1,wherein the step of applying the biological specimen further comprisesapplying the biological specimen to a microscope slide.
 4. The methodaccording to claim 1, wherein the decolorizing step comprises washingthe biological specimen with acid alcohol.
 5. The method according toclaim 1 further comprising the step of applying a counter-staincomposition to the biological specimen after the selected level ofdecolorization is obtained.
 6. The method according to claim 1, whereinthe step of optically analyzing the biological specimen comprises thesteps of: passing radiation of at least one selected frequency throughat least partially stained decolorization fluid after the decolorizationstep; measuring the radiation transmitted through the decolorizationfluid; and comparing the measured transmitted radiation to a thresholddecolorization value.
 7. A method of staining and analyzing biologicalspecimen comprising the steps of: applying the biological specimen to asubstrate; staining the biological specimen with a selected stainingcomposition; at least partially decolorizing the biological specimen bythe application of a decolorizing fluid; collecting the decolorizingfluid; optically analyzing the decolorizing fluid to detect a level ofdecolorization; and repeating, if necessary, the decolorizing step andthe optically detecting step until a selected level of decolorization isobtained.
 8. The method according to claim 7 wherein the stainingcomposition is a Gram's stain composition comprising gentian violet andGram's iodine.
 9. The method according to claim 7, wherein the step ofapplying the biological specimen further comprises applying thebiological specimen to a microscope slide.
 10. The method according toclaim 7, wherein the decolorizing step comprises washing the biologicalspecimen with acid alcohol.
 11. The method according to claim 7 furthercomprising the step of applying a counter-stain composition to thebiological specimen after the selected level of decolorization isobtained.
 12. The method according to claim 7, wherein the step ofoptically analyzing the decolorizing fluid comprises the steps of:passing radiation of at least one selected frequency through at leastpartially stained decolorizing fluid after the decolorization step;measuring the radiation transmitted through the decolorizing fluid; andcomparing the measured transmitted radiation to a thresholddecolorization value.
 13. A method of staining and analyzing biologicalspecimen comprising the steps of: applying the biological specimen to asubstrate; staining the biological specimen with a selected stainingcomposition; at least partially decolorizing the biological specimen;optically detecting a level of decolorization by: passing radiation ofat least one selected frequency through a portion of the biologicalspecimen; measuring the radiation transmitted through the portion of thebiological specimen; and comparing the measured transmitted radiation toa threshold decolorization value.; and repeating, if necessary, thedecolorizing step and the optically detecting step until a selectedlevel of decolorization is obtained.
 14. The method according to claim13 wherein the staining composition is a Gram's stain compositioncomprising gentian violet and Gram's iodine.
 15. The method according toclaim 13, wherein the step of applying the biological specimen furthercomprises applying the biological specimen to a microscope slide. 16.The method according to claim 13, wherein the decolorizing stepcomprises washing the biological specimen with acid alcohol.
 17. Themethod according to claim 13 further comprising the step of applying acounter-stain composition to the biological specimen after the selectedlevel of decolorization is obtained.
 18. The method according to claim13, wherein the portion of the biological specimen is decolorizing fluidfrom the decolorization step.
 19. Apparatus for staining and analyzingbiological specimen comprising the steps of: a substrate carrier forsecuring a substrate having the specimen mounted thereon; a plurality offluid reservoirs for storage and delivery of a plurality of fluids tothe substrate, the fluids including a staining fluid and adecolorization fluid; a collector for collecting decolorization fluidthat runs off the substrate and specimen; a source of radiation arrangedto irradiate decolorization fluid in the collector; a photo detector formeasuring a characteristic of the radiation passing through thedecolorization fluid; and comparison means for comparing the measuredcharacteristic to a selected threshold value indicative of a selecteddecolorization level.